Fate of the replisome following arrest by UV-induced DNA damage in Escherichia coli

Abstract

Accurate replication in the presence of DNA damage is essential to genome stability and viability in all cells. In Escherichia coli, DNA replication forks blocked by UV-induced damage undergo a partial resection and RecF-catalyzed regression before synthesis resumes. These processing events generate distinct structural intermediates on the DNA that can be visualized in vivo using 2D agarose gels. However, the fate and behavior of the stalled replisome remains a central uncharacterized question. Here, we use thermosensitive mutants to show that the replisome's polymerases uncouple and transiently dissociate from the DNA in vivo. Inactivation of α, β, or τ subunits within the replisome is sufficient to signal and induce the RecF-mediated processing events observed following UV damage. By contrast, the helicase-primase complex (DnaB and DnaG) remains critically associated with the fork, leading to a loss of fork integrity, degradation, and aberrant intermediates when disrupted. The results reveal a dynamic replisome, capable of partial disassembly to allow access to the obstruction, while retaining subunits that maintain fork licensing and direct reassembly to the appropriate location after processing has occurred.

Keywords: RecF pathway; replication fork processing.

Fig. 1.

Replication is disrupted by UV-induced damage or following inactivation of the DNA polymerase, τ complex, or helicase–primase complex. (A) A diagram of the replisome, indicating the subunits of each protein complex. (B) Thermosensitive mutants that inactivate the polymerase core, τ complex, or helicase complex are viable at 30 °C but fail to grow at the restrictive temperature of 42 °C following overnight incubation. (C) The rate of DNA synthesis is inhibited following UV-induced damage or inactivation of the replisome’s essential subunits. Wild-type or mutant cultures, grown at 30 °C were pulse-labeled with 1 µCi per 10 µg/mL [³H]thymidine for 2 min at the indicated times following mock treatment (open symbols), 50 J/m² UV irradiation (filled symbols), or a shift to 42 °C (filled symbols). The amount of radioactivity incorporated into the DNA, relative to pretreated cultures is plotted. Error bars represent SE of two experiments.

Fig. 2.

Inactivation of the DNA polymerase or τ complex, but not the helicase complex, induces replication fork processing intermediates similar to UV-induced damage. (A) Diagram depicting the migration pattern of replicating DNA fragments and intermediates associated with processing forks disrupted by UV-induced damage in 2D agarose gels. Strains containing plasmid pBR322 were UV-irradiated with 50 J/m² or filtered and placed in prewarmed media at 42 °C. Genomic and plasmid DNA was then purified, digested with PvuII, and analyzed by 2D agarose gel analysis at 15 min following UV-irradiation and 90 min following temperature shift. (B) In wild-type cultures, replication fork processing intermediates are observed after disruption by UV-irradiation but not after a shift to 42 °C. (C) Disruption of the DNA polymerase core (dnaE^ts), processivity factor (dnaN^ts) or τ (dnaX^ts) is sufficient to induce replication fork processing similar to that seen after UV-induced damage. (D) Abnormal intermediates, distinct from any of those associated with processing UV-induced damage, arise after disruption of the helicase (dnaB^ts) or primase (dnaG^ts). (E) No atypical intermediates are observed following disruption of the nonessential proofreading subunit of the DNA polymerase (dnaQ^ts).

Fig. 3.

Similar to after UV, the integrity of the replication fork DNA is maintained after DNA polymerase dissociation. The integrity of the replication fork is maintained following arrest by UV irradiation or polymerase dissociation, but collapses after disruption of the helicase. [¹⁴C]thymine-prelabeled cultures were pulse-labeled with [³H]thymidine for 20 s before being filtered and placed in nonradioactive media and either UV-irradiated with 50 J/m² or shifted to 42 °C as indicated. The amount of radioactivity remaining in the total genomic DNA (open symbols) and nascent DNA at the replication fork (filled symbols) is plotted over time. Error bars represent SE of two independent experiments.

Fig. 4.

RecF mediates the formation of replication fork intermediates following DNA polymerase dissociation. The formation and processing of the replication intermediates after DNA polymerase dissociation are mediated by RecF, similar to UV. The formation of the abnormal intermediates that arise following helicase disruption is not dependent on RecF. Isogenic wild-type, dnaB^ts, and dnaE^ts cultures containing a normal or mutated copy of the recF gene were UV-irradiated or temperature shifted and analyzed by 2D agarose gel analysis as in Fig. 2.

Fig. 5.

Model of replisome at UV-induced damage. Upon encountering an arresting lesion (PD, pyrimidine dimer) (i), DNA synthesis becomes uncoupled and the polymerases transiently dissociate. (ii) This serves as a signal to initiate the replication fork DNA processing by the RecF-pathway gene products (gray circles) allowing repair enzymes (NER) or translesion polymerases to access the lesion. (iii) The helicase–primase complex remains bound to the template DNA and serves to maintain the licensing and integrity of the replication fork, directing replisome reassembly to the correct location once the lesion has been processed.